Journal of ASTM International, Vol. 8, No. 2 Paper ID JAI103098 Available online at www.astm.org
E. Y. Hashem,1 M. M. Seleim,2 and Ahmed M. El-Zohry3
Spectrophotometric Determination of Iron in Pharmaceutical and Water Samples by Interacting with 5-„4-Nitrophenylazo… Salicylic Acid and Eosin ABSTRACT: A highly stable ternary complex of iron共III兲 with 5-共4-nitrophenylazo兲 salicylic acid 共NPAS兲 and Eosin 共Es兲 in acidic medium at room temperature gave a maximum absorption at 545 nm with a molar absorptivity 2.81⫻104 L mol⫺1 cm⫺1. A spectrophotometric method using these ligands was developed and optimized in terms of pH, stability of the complex, amount of reagent required, sensitivity, linearity, and the effect of various foreign ions was studied. The linear range for iron共III兲 determination is 0.18–6.0 mg L⫺1. The method was sensitive, accurate, and all the reagents were stable under the working conditions. Moreover, the method was easy to perform for the determination of iron in pharmaceutical and water samples. KEYWORDS: spectrophotometry, iron, azo dye, eosin, pharmaceutical, biological, water
Introduction The human body needs daily an amount of vitamins that contain minerals for the biological processes occurring in the body but any vitamin or mineral can be extremely harmful. One of the most abundant minerals in vitamins is iron, its deficiency or large amounts have destructive manners. Excessive iron can be toxic because free ferrous iron reacts with peroxides to produce free radicals, which are highly reactive and can damage deoxyribonucleic acid, proteins, lipids, and other cellular components. Ternary complexes of the general formula 关LnM xSy兴 have been widely used in spectrophotometric analysis 关1–5兴. The study and the analytical applications of ternary complexes attract much interest in pharmaceutical analysis, either in the determination of metal cations, which are present in various formulations and biological samples 关6–8兴. The present study was undertaken to develop a sensitive spectrophotometric method for the determination of iron共III兲, which is the main and essential therapeutic component in various pharmaceutical preparations which are used for the prevention and the treatment of iron-deficiency anaemia. Pervious methods for spectrophotometric determination of Fe共III兲 have some disadvantages like time consuming, low sensitivity, heating process, and the use of large amounts of organic solvents, which could increase the environmental pollution like the following methods: Yoshio shijo 共 = 17.3⫻ 104 L mol−1 cm−1, max = 613 nm, benzene and xylene for extracting, long procedure兲 关9兴, Hemlata Mohabey et al., 共 = 1.21 ⫻ 104 L mol−1 cm−1, max = 520 nm, benzene for extracting兲 关10兴, Lih-Fen 共 = 1.2⫻ 104 L mol−1 cm−1, max = 514 nm, shaking for 10 min in water bath 55° C兲 关11兴, J. Miura 共 = 1.53⫻ 104 L mol−1 cm−1, max = 396 nm兲 关12兴, G.S.R. Krishnamurti 共 = 2.15⫻ 104 L mol−1 cm−1, max = 595 nm兲 关13兴, M.V. Dawson et al. 共 = 2.79⫻ 104 L mol−1 cm−1, max = 562 nm兲 关14兴, Prodromos B. et al., 共 = 2.3 ⫻ 104 L mol−1 cm−1, max = 471 nm, shaking for 30 min at 50° C兲 关15兴, Prodromos B. et al., 共 = 2.08 ⫻ 104 L mol−1 cm−1, max = 386 nm兲 关16兴, N. Hirayama et al., 共 = 0.43⫻ 104 L mol−1 cm−1, max = 560 nm, leave in water bath for 19 min at °C兲 关17兴, M.C.C. Areias et al., 共 = 1.26 ⫻ 104 L mol−1 cm−1, max = 375 nm兲 关18兴, Astrid et al., 共max = 473, 506 nm, chloroform for extracting, shaking for 20 min兲 关19兴. The solution spectra of the mixed ligand complex formed were characterized by high intensity. The study revealed the formation of an mixed ligand complex having the composition Fe共III兲 – NPAS– 共Es兲2. Manuscript received March 24, 2010; accepted for publication November 24, 2010; published online December 2010. 1 Chemistry Dept., Faculty of Science, Assiut Univ., Assiut 71516, Egypt 共Corresponding author兲,
[email protected];
[email protected] 2 Deceased, Chemistry Dept., Faculty of Science, Assiut Univ., Assiut 71516, Egypt. 3 Chemistry Dept., Faculty of Science, Assiut Univ., Assiut 71516, Egypt. Copyright © 2011 by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959.
e-mail:
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The optimum conditions for the spectrophotometric of Fe共III兲 were established. A simple, sensitive, and selective method was proposed for the determination of Fe共III兲. The method was applied successfully to the determination of Fe共III兲 in mineral vitamins and water samples. Experimental Instruments Absorption measurements were measured on a Thermo Evolution 300 recording spectrophotometer using 10 mm matched quartz cells and slit width 2 nm. The pH-meter 共HANNA HI 223兲 equipped with a Radiometer combined glass electrode was used for pH measurements. The pH values in water-ethanol medium were corrected as described elsewhere 关20兴. Chemicals All chemicals were analytical reagent grade and de-ionized water 共or absolute ethanol兲 was used for solutions’ preparations. Standard Procedure Spectrophotometric Determination of Trace Amounts of Fe(III)—The spectrophotometric determination of Fe共III兲 with NPAS and Es employs the complex with molar ratio M : L : A = 1 : 1 : 2. A solution containing 5 g of iron共III兲 共1.0 mL兲 was transferred to 10 mL calibrating flask which contains 1.65 ⫻ 10−3M of NPAS 共1.0 mL兲 and 3.3⫻ 10−3M of Es 共2 mL兲 and 1.0 mL of 1.0M NaClO4 then add the required volume of ethanol to keep the percentage at 30 % ethanol then add the appropriate amount of HClO4 to reach pH of 3.8 and reach to 10 ml as total volume then measure the absorbance against reagent blank at max 545 nm. Absorbance of all solutions investigated remains constant for at least 5 h. Dissolution of Commercial Pharmaceutical Preparations—Commercial pharmaceutical samples were provided from local Egyptian pharmaceutical industries. Ten capsules of each preparation were weighed accurately to determine the average weight of the capsules, the contents of them were grinded to fine powder, dissolved in a mixture of HNO3 / H2SO4 共10:1兲, and heated gently until charring began. Drop wise addition of HNO3 and boiling were continued until a colorless liquid was obtained. A few millilitres of water were added, and the solution was evaporated to evolution of white fumes then it was dissolved in a few millilitres of 0.1M perchloric acid and diluted to 100 mL with bi-distilled water. Results and Discussion Acid-Base Properties of NPAS The absorption spectra of 0.5⫻ 10−4M of the reagent in 50 % ethanol and various concentration of HClO4 and NaOH were recorded as the dependence A = f共兲 for various pH. The ultra violet-visible spectra of NPAS in all mixtures investigated display four absorption bands within all the pH range of 1.4–12.5. The maximum absorption of these bands were located at 365, 380, 390, and 520 nm. The different acid-base equlibria existing in solution of NPAS may be represented by the following equations 关21兴: −H+ −H+ −H+ + − LH3 LH2 LH L2− 共6.0–9.3兲 共9.5–11.5兲 共3.7–5.5兲 共1.4–3.1兲
max = 365 nm
max = 380 nm
max = 390 nm
max = 520 nm
Absorption Spectra and Optimum pH The solution spectra of NAPS exhibited a main absorption band at = 365 nm at pH range of 1.4–3.1. The spectra of Fe共III兲–NAPS 共1:1兲 complex with the reagent blank as reference were characterized by an
Absorbance
EL-ZOHRY AND HASHEM ON SPECTROPHOTOMETRIC DETERMINATION OF IRON 3
1.2
6
1.0
7
0.8
5
4
0.6
3 2
0.4
8
0.2
1
0.0 525
550
9 575
600
625
650
675
Wavelength(nm) FIG. 1—Absorption for Fe(III)-NPAS-Eosin complexes, 30 % ethanol, I ⫽ 0.1M NaClO4, CNPAS ⫽ CFe共III兲 ⫽ 0.66 ⫻ 10⫺4M, CEosin ⫽ 1.33 ⫻ 10⫺4M, pH; 1—0.85, 2—1.0, 3—1.5, 4—1.7, 5—2.7, 6—4.3, 7—4.8, 8—5.9, and 9—7.8. absorption maximum at max 416 nm in the pH of 1.5–2.5, similar behaviour was observed for the spectrum of the Fe共III兲–Es complex, neither Es nor its 1:1 complex with Fe共III兲 had a measurable absorbance above 250 nm. The effect of pH in the range 1.0–6.0 on the spectrum of solutions containing equimolar concentrations of the NPAS, Es, and Fe共III兲 against a reagent blank containing the same concentrations of the two ligands showed an absorption band with max at 545 nm at pH 3.8 共Figs. 1 and 2兲. This band was unambiguously due to the formation of the mixed ligand complex of Fe共III兲 with NAPS and Es. The solution spectra of the ternary system in a 1:1:2 molar ratio and also the spectra of Fe共III兲
1.2
Absorbance
1.0
0.8
0.6
0.4
0.2 1
2
3
4
5
6
pH FIG. 2—Variation of absorbance with pH for Fe(III)–NPAS–Eosin system at max ⫽ 545 nm, CNPAS ⫽ CFe共III兲 ⫽ 0.66 ⫻ 10⫺4M, CEosin ⫽ 1.33 ⫻ 10⫺4M.
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1.5
Absorbance
Ternary Fe(III)-NPAS-Eosin Eosin
NPAS. 1.0
Binary Fe(III)-NPAS
0.5
300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675
Wavelength (nm) FIG. 3—Absorption spectra of different constituents of the ternary complex at pH 3.8, CNPAS ⫽ CEs ⫽ 0.5 ⫻ 10⫺4M, CFe共III兲 ⫽ 1 ⫻ 10⫺4M. complex with NAPS and Es in 30 % 共v/v兲 ethanol with reagent blank as reference are given in Fig. 3. The formation of the mixed complex was investigated at different pH values using equimolar concentrations of components, the absorbance versus pH graphs at 545 nm indicate the existence of two basic equilibria that are sufficiently separated. The analysis of the two rising parts of these graphs within the pH ranges were performed assuming the following scheme: Fe3+ + H3L+ ⇔ FeLH2+ + 2H+
共1兲
FeLH2+ + 2A− ⇔ 关FeLHA2兴 共1兲
共2兲
共2兲
H3L+ and A symbolizing the molecular forms of NAPS and Es, respectively. The stability constants of the ternary complex Fe共NAPS兲共Es兲2 was related to the equilibrium constant K by the expression 共Table 1兲. We can see that the value of stability constant is high due to the high stability of the complex. Composition of the Fe(III) Ternary Complex Job’s method of continuous variation was applied to establish the composition of the ternary Fe共III兲 NAPS and Es complex the molar fractions of two of the components were varied continuously, keeping the third component in a large excess for all solutions in the series 共Figs. 4–7兲. The obtained results indicated that TABLE 1—Mean values of equilibrium constant 共log Kⴱ兲, stability constants 共log 兲, and molar absorptivity 共兲 for Fe–NPAS–Es. Equilibriuma FeLH+ 2 Es FeLH共Es兲2 FeLH共Es兲2 关Fe兴关LH3兴 + 关Es兴2
Constant Kd 1
Log Constant 共0.84兲b 共15.1兲c
Note: Complexes 关I = 0.1M共NaClO4兲, 25° C, 30 % 共V/V兲 ethanol. a Charges are omitted. b From the absorbance versus pH graphs for solutions of equimolar concentrations. c log  = log Kⴱ + pK1 + log binary. d K = formation constant;  = stability constant.
Molar Absorptivity 共兲L mol− cm−1 2.81⫻ 104
EL-ZOHRY AND HASHEM ON SPECTROPHOTOMETRIC DETERMINATION OF IRON 5 1.8 1.6 1.4
3
4
Absorbance
1.2
5
1.0
2
0.8 0.6 0.4 0.2
1
0.0 550
575
600
625
650
675
700
Wavelength(nm) FIG. 4—Continuous variation for Fe⫹3 ternary complex at max ⫽ 545 nm, CFe共III兲 ⫽ 0.66 ⫻ 10⫺4M, CEosin ⫽ 1.33 ⫻ 10⫺4, pH ⫽ 3.8, NaClO4 ⫽ 0.1M, 30 % ethanol, CNPAS; 1—0.33 ⫻ 10⫺4, 2—0.5 ⫻ 10⫺4, 3—0.66 ⫻ 10⫺4, 4—0.83 ⫻ 10⫺4, and 5—1 ⫻ 10⫺4M. the overall Fe共III兲 complex had a 1:1:2 compositions at the pH of the study. The stoichiometry of the ternary system was also determined by applying the molar ratio method 关22,23兴. Calibration Graph and Reproducibility At the optimum conditions given under the standard procedure a linear calibration graph is obtained up to 0.18 g mL−1 of Fe共III兲. A ring boom plot showed that the optimum range for the determination of Fe共III兲 1.4 1.2
Absorbance
1.0 0.8 0.6 0.4 0.2 0.0 0.1
0.2
0.3
0.4
0.5
+3
+3
0.6
0.7
0.8
0.9
[Fe ]/([Fe ]+[NPAS]) FIG. 5—Continuous variation for Fe⫹3 ternary complex at max ⫽ 545 nm, CEosin ⫽ 1.33 ⫻ 10⫺4, pH ⫽ 3.8, NaClO4 ⫽ 0.1M, 30 % ethanol.
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3
1.4
Absorbance
1.2
4 5
1.0 0.8 0.6
2
0.4
1
0.2 0.0 525
550
575
600
625
650
675
700
Wavelength (nm) FIG. 6—Continuous variation for Fe⫹3 ternary complex at max ⫽ 545 nm, CFe共III兲 ⫽ 0.5 ⫻ 10⫺4M, CNPAS ⫽ 0.66 ⫻ 10⫺4, pH ⫽ 3.8, NaClO4 ⫽ 0.1M, 30 % ethanol, CEosin; 1—1.66 ⫻ 10⫺4, 2—1.33 ⫻ 10⫺4, 3—1 ⫻ 10⫺4, 4—0.66 ⫻ 10⫺4, and 5—0.33 ⫻ 10⫺4M.
is 0.3– 6.0 g mL−1. The sensitivity of reaction was calculated according to Sandell and was founded to be 1.95⫻ 10−3 g cm−2 of Fe共III兲. The reproducibility of the method was checked by analyzing two series of five solutions having Fe共III兲 concentrations of 0.5 and 2.0 g mL−1. The relative standard deviations were found to be 0.59 and 0.44 % receptivity. The detection limit for Fe共III兲 was found to be 5.0 ng mL−1 共Table 2兲.
1.4 1.2
Absorbance
1.0 0.8 0.6 0.4 0.2 0.0 0.0
0.1
0.2
0.3
0.4 +3
0.5
0.6
0.7
0.8
0.9
+3
[Fe ]/([Fe ]+[Eosin]) FIG. 7—Continuous variation for Fe⫹3 ternary complex at max ⫽ 545 nm, CNPAS ⫽ 0.66 ⫻ 10⫺4, pH ⫽ 3.8, NaClO4 ⫽ 0.1M, 30 % ethanol.
EL-ZOHRY AND HASHEM ON SPECTROPHOTOMETRIC DETERMINATION OF IRON 7 TABLE 2—Conditions for the spectrophotometric determination of iron(III). Wavelength of maximum absorbance 共nm兲 Limit of detection 共LOD兲 共g / mL兲 Range of linearity 共g / mL兲 Molar absorptivity 共L mol−1 cm−1兲 Optimum pH Correlation coefficient 共r兲 Relative standard deviation 共%兲
545 nm 5.0⫻ 10−3 0.3–6.0 2.81⫻ 104 3.5 0.991 0.44–0.59
Effect of Diverse Ions The effect of diverse ions on the determination of Fe共III兲 at the 2 g mL−1 level was investigated. The criteria for interference were an absorbance value varying by more than ⫾2 % from the expected value for Fe共III兲. The determination of Fe共III兲 as FeLA2 is possible in the presence of NH+4 , Cl−, Br−, I−, NO−3 , ClO−4 , 3+ 2+ 2+ acetate, EDTA, sulphate, citrate, borate, Ca2+, Zn2+, Hg2+, Ag+, MoO2− 共up to 200 fold 4 , Al , Ba , Sr 2+ 2+ 2+ 2+ 2+ 2+ excess兲, Ni , Co , Cu , Mn , Sn , and Cd 共up to 120 fold excess兲. The alkali metal ions have no interfering effect on the determination of iron共III兲. Applications To confirm the usefulness of the proposed method, Fe共III兲 had been determined simultaneously in four pharmaceuticals vitamins samples 共Table 3兲. The results obtained were in good agreement with the certified values of pharmaceuticals vitamins and water samples the RSD value was found in the range 0.43– 0.59 % for iron共III兲 in all pharmaceuticals vitamins and water samples. Conclusions The numbers of used reagents in the iron determination in aqueous media were limited and most of them require a chromogen for spectrophotometric methods. The ligand which was used in this study had several advantages over other ligands currently utilized for photometric iron determination and it could be obtained at high yields by a simple method. This reagent forms highly stable 1:1:2 complexes with Fe共III兲 in 30 % ethanol media, at max 545 and molar absorptivity 2.81⫻ 104 L mol−1 cm−1. Our method could be easily applied to various pharmaceutical, environmental, and biological samples for determination of 0.3–6.0 mg/L iron. Moreover, the method is reproducible and precise as seen from the obtained results. Therefore, the method could be regarded as rapid, simple, sensitive, reproducible, and also appropriate for direct determination of iron.
TABLE 3—Determination of iron(III) in multi-vitamins. Sample Commercial Namec Vitona plus Megamine Max vit. Kerovit Water samples Tap water Mineral water
Certified Valuea 关Fe共III兲兴 140 37.5 3.0 90
Amount Found by AAS 共%兲 关Fe共III兲兴 139.7 37.51 2.99 90
Amount Found by Proposed Method 关Fe共III兲兴 139.85 37.5 2.98 90.05
R.S.D.b 共%兲 关Fe共III兲兴 0.44 0.52 0.59 0.43
Recovery 共%兲 关Fe共III兲兴 99.89 100.0 99.33 100.05
14.70d 2.86d
14.81d 2.90d
14.72d 2.88d
0.46 0.58
100.13 100.69
Note: AAS⫽atomic absorption spectroscopy. a Amount in 10 capsules 共mg兲. b N=5 c Producers: Amoun, Egyptian International Pharmaceutical Industries Co., Nature’s Life. d g L−1.
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